Pain and Analgesia (A*) Flashcards

Question

A*: Describe the course and distribution of primary afferent neurones in the dorsal horn of the spinal cord.

Answer 1

- Primary afferent neurones enter the white matter of the dorsal horn via the dorsal roots. - Once in the dorsal horn, axons carrying pain and temperature information form the dorsolateral tract of Lissauer, which comprises both ascending and descending collaterals. - These collaterals travel 1-2 spinal levels (up or down) and enter the grey matter of the dorsal horn. - In the grey matter, the collaterals synapse with second-order neurones in Rexed laminae I and II (the two most ventral grey matter regions): 1 - Rexed lamina I is the marginal zone. 2 - Rexed lamina II is the substantia gelatinosa. - Information carried by second-order neurones in the substantia gelatinosa is carried further to Rexed laminae IV, V and VI, known collectively as the nucleus proprius. The nucleus proprius also receives additional innervation from the first-order neurones directly. - Second-order neurones from Rexed laminae I-VI (marginal zone, substantia gelatinosa and the nucleus proprius) ascend the spinal cord to the thalamus as the spinothalamic tract (AKA anterolateral system).

Answer 2

Mechanisms by which NGF contributes to chronic pain: - NGF is signalling is elevated in chronic pain (and also following injury). This contributes to chronic pain because: 1 - NGF upregulates genes that code for proteins that potentiate the pain response, such as: - Substance P. - TRPV1. - Nav1.8 and 1.9 Na+ channels. - NGF was found to be upregulated in the DRG and PAG in rat models of pain (Violi et al., 2010). 2 - NGF promotes axon sprouting. - Immediately following axotomy, NGF is unable to reach the target tissue, and therefore declines. - In response to this decline, pain-activated glia, namely satellite cells, release an overcompensation of NGF. - This promotes induction of sprouting following axotomy, leading to neuroma formation and spinal cord reorganisation. - A potential analgesic drug targeting NGF is acetyl-L-carnitine (ALCAR), which was shown to reduce NGF to physiological levels in the Violi et al. paper. - Anti-NGF antibodies are also useful in the animal models of pain which are insensitive to the analgesic effects of opioids and NSAIDs (da Silva et al., 2019). NGF-targeting drugs might be a potentially useful drug in treatment-resistant chronic pain, however the ubiquity of NGF in the CNS and PNS means that there will likely be many adverse effects of anti-NGF treatment. - However, it has also been shown that NGF can decrease pain transmission by increasing expression of BDNF in the DRG, which in turn facilitates GABA release. Hence, it is probably not as simple as 'NGF increases pain'.

Answer 3

- NSAIDs inhibit COX enzymes, resulting in a decrease in prostaglandin expression. - Prostaglandins are involved in the induction of inflammatory responses. - By reducing inflammation, fewer inflammatory substances (e.g. histamine, H+, prostaglandins, TNF-alpha, bradykinin and cytokines) are available to sensitise nociceptors. * See card 12.

Answer 4

- Bradykinin can cause pain by activating phospholipase A2, which produces arachidonic acid from membrane lipids. - This results in an increase in production of prostaglandins, promoting inflammation (a positive feedback loop since bradykinin is a product of inflammation).

Answer 5

- Acid-sensing ion channel 3 (ASIC3) is a proton-gated ion channel involved in chemosensation and mechanosensation. - Acidosis can be induced by tissue injury and inflammation. - Activation of ASICs in response to acidosis causes an inward current of Na+, Ca2+ and K+, generating excitatory postsynaptic potentials in peripheral nociceptors. - ASIC knockout mice have been shown to have a reduced response to tissue acidosis-induced hyperalgesia (Deval et al., 2008). - ASIC inhibitors have potential for use in pain induced by tissue injury (not neuropathic pain), e.g. in inflammatory conditions (e.g. infection, rheumatic disorders, lupus and MS). - Black mamba venom was found to contain ASIC blockers known as mambalgins, which have the potential to be used as potent analgesics (Diochot et al., 2012). * See A8 card 35 for TRPV1.

Answer 6

Background: - CB receptors involved in the modulation of pain are found mostly on Adelta fibres, but also on some C fibres. - Noxious stimuli increase endocannabinoid release. - CB1 receptors are located both centrally and peripherally in immune cells. - CB2 receptors are located peripherally in immune cells. - Both CB receptors are Gi/o GPCRs. Analgesia mechanism: - CB1 receptors can attenuate synaptic transmission of ascending pain pathways (through the downstream effects of Gi/o). - Both CB1 and CB2 receptors are expressed in various inflammatory cells, and have an analgesic effect in inflammatory hyperalgesia. - This effect is through attenuating NGF-induced degranulation of mast cells, reducing inflammation-induced peripheral sensitisation. - Therapeutically, CB1 agonists are not preferable because of the side effects associated with the modulation of central synaptic transmission. - Selective CB2 agonists are a potential avenue for analgesic drugs. - Tolerance and dependence are potential issues with these treatments.

Answer 7

- Glia do not directly mediate pain, but can enhance existing pain signals. - Glia can be activated by a variety of neuronal factors, such as those released in the inflammatory soup (card 13) and paradoxically by opioids. 4 successive glial activation states have been reported in chronic pain (Ji et al., 2013): 1 - 'Glial reaction'. - Upregulation of mitogens. - Hypertrophy. - Proliferation. 2 - Activation of MAPK signalling, e.g. ERK, due to the activity of mitogens which are upregulated in the glial reaction. - It has been shown that ERK signalling mediates central sensitisation in dorsal horn neurones, because ERK regulates glutamate and K+ transmission. 3 - Increased expression of ATP, intercellular connexin channels (AKA hemichannels) and chemokine receptors, and decreased expression of glutamate transporters. - ATP influences pain signalling via P2Y receptors on neurones and glia. - Hemichannels permit flow of small signalling molecules such as ATP, ions and microRNAs. - Chemokines attract inflammatory cells, promoting an inflammatory response. - Glutamate transporters are required for glutamate uptake from the synapse. This is an important process in the cessation of signal generation at glutamatergic synapses. Hence, reduced expression of glutamate transporters prolongs glutamate-induced pain signals. 4 - Increased expression of growth factors (e.g. NGF - card 26), chemokines, cytokines and other glial mediators. - These changes (1-4) alter neurone-glial and glial-neurone communication, and hence contribute to chronic pain by causing both central and peripheral sensitisation. Analgesic drugs that inhibit glial activation include: 1 - Propentofylline. 2 - Minocycline.

Answer 8

- It is generally thought that the analgesic effect of gabapentin is due to inhibition of voltage-gated Ca2+ channels, preventing excitatory transmission in ascending pain pathways. - This has been evidenced, for example, by the fact that gabapentin inhibits neurotransmitter release in neurones in vitro. - Voltage-gated Ca2+ channels have 5 subunits: alpha 1, alpha 2, beta, gamma and delta. The alpha 2 and delta subunits are connected with a disulfide bond. - The putative binding site of gabapentin is on the alpha 2-delta subunit of voltage-gated Ca2+ channels. - There is also evidence for binding sites on Na+ leak channels and NMDARs. * Gabapentin is an antiepileptic drug, so the mechanism of action reflects its ability to attenuate synchronous firing seen in epilepsy.

Answer 9

- When second-order neurones ascend the pain pathway, they make one synapse in the medulla and one in the thalamus: 1 - In the thalamus, the second-order neurones synapse with neurones that make connections with enkephalin-releasing neurones in the PAG. 2 - In the medulla, the second-order neurone synapses with enkephalin-releasing neurones in the nucleus reticularis paragigantocellularis (NRPG). In both cases: - The enkephalin-releasing neurones synapse with GABAergic neurones in the PAG, causing inhibition. - The GABA neurones normally tonically inhibit glutamatergic neurones, so loss of the GABAergic inhibition means an increase in firing of the glutamatergic neurones. - These glutamatergic neurones make excitatory descending connections with enkephalin-releasing neurones in the nucleus raphe magnus (NRM) in the medulla. - These enkephalin-releasing neurones, like in the PAG and NRPG, inhibit GABAergic neurones. - As before, the GABAergic neurones in the medulla normally tonically inhibit 5-HT neurones, so loss of the GABAergic inhibition means an increase in firing of the 5-HT neurones. - These 5-HT neurones make excitatory descending connections through the dorsolateral fasciculus with enkephalin neurones in the dorsal horn, which in turn inhibit both the first-order and second-order ascending sensory neurones. - Also, noradrenergic neurones from the locus coeruleus in the pons make descending connections with second-order sensory neurones, causing inhibition. - Descending serotonergic facilitation is a form of central sensitisation present in chronic pain in which serotonergic neurones are pathologically hypoactive. This hypoactivity results in loss of inhibition of ascending pain signals by reducing activity of enkephalin-releasing neurones in the dorsal horn. 1 - Opioids inhibit nociceptor stimulation in the periphery. 2 - Opioids stimulate enkephalin-releasing neurones in the NRPG and PAG. 3 - Opioids inhibit enkephalin-releasing neurones in the dorsal horn.

Answer 10

Drugs targeting the opioid system to achieve analgesia: 1 - Morphine, an opioid receptor agonist. 2 - Codeine, an opioid receptor agonist. 3 - Nalorphine, a partial agonist for the mu opioid receptor. 4 - RB120, an enkephalinase inhibitor. Problems with opioid analgesics: 1 - Opioids can cause dependence. This is due to the presence of opioid receptors in the VTA-NAc neurones, stimulating the mesolimbic reward pathway. - Methadone is an opioid agonist at these receptors that can be used to treat opioid dependence. It is preferable because it does not produce euphoria, and it has a long-lasting action. 2 - Opioids cause respiratory depression. This is due to the presence of opioid receptors in the respiratory centres in the brainstem. - One way of countering this clinically is with 5-HT4A agonists. 5-HT4A receptors have an opposing effect to opioid receptors in respiratory centres, but they do not oppose the analgesic effects of opioid receptors on the descending pain pathway.

Answer 11

Structure of TRPV1: - TRPV1 has 6 transmembrane domains. - The channels generally arrange as either homomeric or heteromeric tetramers. - The transmembrane segment between domains 5 and 6 of each subunit contributes to the ion pore. Role of TRPV1 channels in pain: Summary of points from cards above: 1 - Phosphorylation of TRPV1 in response to components of the inflammatory soup is part of the process of peripheral sensitisation in response to neurogenic inflammation. 2 - TRPV1 antagonists are a novel analgesic drug class. 3 (A*) - TRPV1 is upregulated by NGF, which is overexpressed in pain states. New A* points: - TRPV1 is involved in chemosensation and thermosensation. 1 - Chemosensation. - TRPV1 responds to a number of chemical stimuli and toxins, for example capsaicin, allicin (a protein derived from garlic and onions) and spider toxins, and inflammatory stimuli, such as factors in the neurotrophic soup (see card 12 for a list). - There is also evidence to suggest that TRPV1 responds to lactate, the substance that mediates acidosis following ischaemia. However, the role of TRPV1 in acidosis-induced pain is unclear: - Evidence for the role of TRPV1 channels in the sensation of acidosis is abundant. For example, it has been demonstrated that lactate potentiates CGRP release from the spinal cord in mice (Wang and Fiscus, 1997), a pronociceptive signal. Since CGRP release is generally considered to be a TRPV1-dependent process, this finding would imply that lactate increases TRPV1 activity, resulting in increased nociception. Furthermore, acidotic conditions have been shown to cause a shift in the temperature-response relationship of TRPV1, resulting in sensitisation to inflammatory stimuli. - However, more recent evidence by de la Roche et al. (2016) investigating the functions of the TRPV1 channel more directly found that lactate inhibits TRPV1. This study found that TRPV1 receptor potentials and TRPV1-induced Ca2+ influx are attenuated by addition of lactate. - Hence, there is a need to contextualise the role of TRPV1 signalling in acidosis-induced pain, considering the interaction with other signalling cascades such as ASIC3 (see A* card 29), because although there is abundant evidence to suggest that TRPV1 channels are involved in acidosis, the mechanisms by which signal transduction occurs is unclear. 2 - Thermosensation. - Thermosensation is a more putative function of TRP channels. It is mediated by a range of TRP channels. Specifically, TRPV1 channels are hot (>45 degrees celsius) polymodal nociceptors, TRPV3/4 receptors are hot (>45 degrees celsius) cutaneous thermoreceptors and TRPM8 channels are cold (<15 degrees celsius) cutaneous thermoreceptors. - Thermosensation information is carried by C fibres. - The C-terminus of the TRPV1 and TRPM8 channels (at the distal end of S6) is thought to be responsible for their heat-sensing properties. This is evidenced by a study by Brauchi et al., in which the C-termini of TRPV1 and TRPM8 channels were swapped, resulting in cold-sensing TRPV1 channels and heat-sensing TRPV1 channels. - The ability of thermosensing TRP channels to detect changes in temperature lies in their increased susceptibility to changes in enthalpy compared to other sensory receptors. That is, they are able to more efficiently absorb thermal energy at a constant pressure. This thermal energy is converted into a conformational change, which in turn modifies the probability of depolarisation. Potential TRPV1-targeting drugs: - Counterintuitively, topical application of capsaicin-containing creams was shown to produce an analgesic effect in rheumatoid arthritis. This is likely due to the ability of capsaicin to desensitise TRPV1 channels. However, the cream produces a burning sensation immediately following application, presumably due to the acute activation of TRPV1 channels prior to desensitisation. - Most TRPV1-targeting analgesics are TRPV1 antagonists. The majority of TRPV1 antagonists are developed by modification of capsaicin, and hence are primarily competitive agonists of the capsaicin-binding site. One such drug is capsazepine, which did not see clinical application due to its poor bioavailability and low specificity for TRPV1 channels. Many TRPV1 antagonists followed, showing improved selectivity and more favourable pharmacokinetic properties. A number of TRPV1 antagonists are now in clinical trials. - Another emerging drug class of TRPV1-targeting drugs are voltage-dependent open-channel TRPV1 blockers such as ruthenium red. They arguably have greater clinical potential than orthosteric antagonists since they are able to selectively target overactive TRPV1 channels. Hence, open channel blockers have a greater potential for blocking TRPV1 action whilst producing minimal adverse effects since they do not block nonpathological TRPV1 activation . However, like capsazepine, ruthenium red shows poor specificity for TRPV1 channels, diminishing the benefit of being an open-channel blocker. There is therefore demand for developing open-channel blockers with greater specificity than ruthenium red.

Answer 12

Subtypes of Na+ channels whose primary function is in pain signalling include Nav1.3, 1.7, 1.8 and 1.9. - Nav1.7 is primarily expressed in the peripheral branches of DRG neurones, and respond to EPSPs generated by nociceptors at the nerve terminals, amplifying the generator potential. Nav1.7 channels have a relatively hyperpolarised voltage-dependence of activation. Hence, Nav1.7 channels set the pain threshold because the generator potential quickly exceeds the Nav1.7 threshold potential. Like Nav1.3 TTX-resistant channels, and unlike Nav1.8 and 1.9 channels, Nav1.7 channels tend to accumulate in neuromas, contributing to ectopic depolarisation following nerve injury. The role of Nav1.7 channels in pain is evidenced, for example, by the fact that gain-of-function mutations to Nav1.7 channels have been linked to pain disorders such as inherited erythromelalgia and paroxysmal extreme pain disorder (Waxman and Zamponi, 2014). Inherited erythromelalgia is an autosomal dominant condition that is characterised by episodes of burning pain in the extremities. The gain-of-function mutation in inherited erythromelalgia results in a decreased threshold potential, facilitating pain transmission. In paroxysmal extreme pain disorder, a similar gain-of-function mutation results in severe pain in the rectum and face in response to normally nonpainful stimuli. Selective Nav1.7 antagonists have been shown to significantly reduce pain in inherited erythromelalgia in phase 2 clinical trials. Antiepileptics such as carbamazepine have also shown efficacy for the symptomatic treatment of inherited erythromelalgia, however this also results in side effects of nonselective VGSC channel blockade, such as nausea, dizziness and somnolence, that are not present with selective Nav1.7 blockers. On the other hand, expression of Nav1.7 channels is mostly restricted to DRG neurones, making diverse side-effects unlikely, with the most likely contender being anosmia due to the role of Nav1.7 channels in olfaction. - Like Nav1.7, Nav1.8 is primarily expressed in the peripheral branches of DRG neurones. However, their voltage-dependence of activation is relatively depolarised compared to Nav1.7, (i.e. the threshold potential of Nav1.8 channels is more positive than Nav1.7). Hence, in the rising phase of depolarisation, Nav1.8 channels are responsible for a significant proportion of the total sodium current. There is less evidence for the involvement of Nav1.8 in chronic pain, however gain-of-function mutations are found in small numbers of patients with peripheral neuropathy where Nav1.,7 mutations are absent (Faber et al., 2012). Therefore, Nav1.8 blockade might prove useful in patients that do not respond to Nav1.7 blockade. Numerous Nav1.8 blockers are currently in preclinical stages, and have demonstrated efficacy in rodent models of neuropathic pain. - The Nav1.9 channel is also expressed in the peripheral branches of DRG neurones, but is also found in the myenteric plexus. Like Nav1.7 channels, Nav1.9 channels have a relatively hyperpolarised voltage-dependence of activation. Furthermore, these channels induce slow inward Na+ currents, and therefore do not contribute significantly to the rising phase of depolarisation. Rather, Nav1.9 channels play a role in extending the pain signal and enhancing excitability. It is believed that Nav1.9 channels play a role in mediating inflammatory pain, since knockout of these channels in mice results in decreased pain response to inflammatory stimuli. No Nav1.9 blockers have yet seen preclinical trials, but may show promise for treating pain in chronic inflammatory conditions such as rheumatoid arthritis, lupus and multiple sclerosis. However, Nav1.9 blockers would be expected to pose a potential risk for GIT side effects due to the involvement of Nav1.9 channels in the enteric nervous system and regulation of GIT function. Hence, if Nav1.9 blockers see clinical trials, the analgesic effect of Nav1.9 blockade will have to be sufficiently large so as to justify foregoing the reduced side effects of Nav1.7 blockade. - Nav1.3 channels are not expressed in physiological conditions. However, expression of Nav1,3 channels increases in neuromas in response to disturbed peripheral availability of NGF. Nav1.3 has similar kinetics to Nav1.7 in that it has a relatively hyperpolarised voltage-dependence of activation, serving to amplify generator potentials. However, the ability to rapidly repolarise makes Nav1.3 channels able to carry out repetitive firing. Nav1.3 blockers have been in development for a number of years, however very few have demonstrated pharmacokinetic properties that are suitable for clinical use. Development of Nav1.3 blockers might prove useful for the treatment of peripheral nerve injury. Since Nav1.3 expression is limited to pathological conditions, it would not be expected that Nav1.3 blockade would produce any side effects, making them clinically attractive targets.

Answer 13

- Astrocytic ATP is converted by ATPases in the extracellular space into adenosine. - Adenosine binds to A1 receptors in neurones in the ascending pain pathways. - A1 receptors are Gi/o coupled, and therefore cause inhibition when activated. - However, there is also evidence of the involvement of other gliotransmitters in pain modulation, as pain threshold was reduced in mice underexpressing glial SNARE proteins (Foley et al.,2011). - This suggests involvement of other gliotransmitters requiring Ca2+dependent transport, such as glutamate, GABA or D-serine, in potentiating pain transmission. - This study was met with controversy over the exclusivity of the targeted SNARE protein to astrocytes. However, the targeted SNARE protein was shown to colocalise with the astrocyte-specific protein, GFAP, but not the neuronal-specific protein, NeuN, or microglial-specific protein, Iba1. These are widely used and well-validated markers with high specificity to these cell types, hence the SNARE protein was likely astrocyte-specific.

Answer 14

- Substance P and CGRP bind to NK-1 receptors in pain fibres. - Binding to NK-1 receptors causes PKC-mediated NMDA receptor phosphorylation. - This increases the affinity of glycine for the glycine site on the NMDA receptor, potentiating NMDA-mediated transmission of pain signals in primary afferent neurones. - This only occurs when sufficient glutamate is coreleased with the neuropeptide, because sufficient AMPA receptors must first be activated in order to overcome the voltage-dependent Mg2+ block of the NMDA receptors for the neuropeptides to have any effect on transmission. - NAAG is coreleased with these neuropeptides, which has the ability to bind to presynaptic mGlu3 receptors as a means of negative feedback (NAAG has an analgesic effect). ---------------------- - Nociceptin and nocistatin are opioid-related neuropeptides that are produced from the same prepropeptide. - Nocistatin is not biologically active on its own but can antagonise the effects of nociceptin. - Nociceptin (considered an anti-opioid) causes allodynia, and is therefore a potential target for pain therapy. ---------------------- - Many neuropeptidergic neurones exhibit strong neuroplasticity. - In the dorsal root ganglion (DRG), the phenotype of neurones is influenced by peripheral nerve lesions. - For example, galanin and is upregulated in the DRG in response to nerve injury. - Galanin has analgesic effects at Gal1 receptors and pronociceptive effects at Gal2 receptors.